SUMMARY Methods and techniques capable of directly visualizing chemical bonding and charge in biomolecules would add an exciting tool to the field of structural biology. Unlike X-rays, electron diffraction is very sensitive to the bond- charge distribution, especially at lower resolution and small scattering angles, so that electron diffraction has been used on inorganic samples to study and image the charge distribution in chemical bonds, directly mapping out both covalent and ionic bonds. This sensitivity is readily apparent from the large difference between tabulated electron scattering factors for atoms and those for ions. The overall aim of this project is to develop and extend charge-cloud modeling methods to biomolecular structures and couple these procedures with microcrystal electron diffraction (MicroED) data collection and processing for the direct determination of chemical bonding in high-resolution biomolecular structures. The charge-cloud method was initially developed to determine the bonding in an organic small molecule phthalocyanine and used transmission electron diffraction patterns from thin crystals. It was shown that by properly treating electron diffraction data with this method, charge density in the bonds between atoms could be seen in a 2D projection of the organic molecular structure. To extend this method to 3D structures of biological molecules, high-quality 3D electron diffraction data will be required. Therefore, we will make use of the MicroED method for electron diffraction data collection on several model microcrystalline samples. Since its development, MicroED has been shown to produce high-quality electron diffraction data that yields high-resolution 3D structures of biological molecules. The combined method of MicroED data collection and processing along with charge-cloud refinement of low order Bragg reflections will yield a straightforward, efficient, widely accessible technique for the direct visualization of bonding in biological samples.